Reagent kit for primary culture on umbilical cord mesenchymal stem cells

A technology of mesenchymal stem cells and primary culture, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems that affect the research and clinical application of umbilical cord MSCs, the complex composition of fetal bovine serum, and the contamination of exogenous viruses , to achieve good wall-attachment effect, conducive to growth and proliferation, and to promote the effect of proliferative activity

Inactive Publication Date: 2017-08-11
济南赛尔生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fetal bovine serum has complex components, is unstable between batches, is not easy to control quality, is easy to introduce foreign virus contamination, and the cultured cells are easy to age, and the cost is also high
Although there are serum-free media on the market, there are problems of slow primary proliferation, small number of cells, and easy aging, which seriously affect the research and clinical application of umbilical cord MSCs

Method used

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  • Reagent kit for primary culture on umbilical cord mesenchymal stem cells
  • Reagent kit for primary culture on umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: the preparation of kit one

[0022] 1. Umbilical cord washing liquid: take 99 parts by volume (unit: ml) of normal saline, and 1 part by volume (unit: ml) of 100× penicillin-streptomycin solution (final concentrations are 100 U / ml and 100 μg / ml respectively) , mix well. Store at room temperature after preparation, and prepare immediately for use.

[0023] 2. Coating solution: Weigh 0.5g of fibronectin and dissolve it in 100ml of normal saline, fully dissolve, filter and sterilize at 0.22μm, aliquot, and store at 4°C. Before cell plating, each T75cm 2 Add 5ml of the prepared 0.5g / 100ml fibronectin to the flask, shake the flask gently so that the solution covers the bottom of the flask, and put it in 37°C, 5% CO 2 Place in the incubator for 30-60 minutes. 5 minutes before cell inoculation, the coating solution of the culture bottle was aspirated and discarded.

[0024] 3. Primary cell culture medium:

[0025] (1) Dissolve human insulin, transferrin, hu...

Embodiment 2

[0027] Embodiment 2: the preparation of kit two

[0028] 1. Umbilical cord washing solution: 99 parts by volume (unit: ml) of PBS buffer solution at pH 7.4, 100× penicillin-streptomycin solution (final concentrations of 100 U / ml and 100μg / ml), mix well. Store at room temperature after preparation, and prepare immediately for use.

[0029] 2. Coating solution: Weigh 0.2g type IV collagen and dissolve it in 100ml normal saline, fully dissolve, filter and sterilize at 0.22μm, aliquot and store at 4°C. Before cell plating, each T75cm 2 Add 5ml of the prepared 0.2g / 100ml type IV collagen to the flask, shake the flask gently to make the solution cover the bottom of the flask, and put it in 37℃, 5% CO 2 Place in the incubator for 30-60 minutes. 5 minutes before cell inoculation, the coating solution of the culture bottle was aspirated and discarded.

[0030] 3. Primary cell culture medium:

[0031] (1) Dissolve human insulin, transferrin, human serum albumin, vitamin C, trehalo...

Embodiment 3

[0033] Example 3: Primary culture of umbilical cord mesenchymal stem cells

[0034] 1. Take three new T75cm 2 For the culture bottle, one bottle is coated with the coating solution of Kit 1, marked as A; one bottle is coated with the coating solution of Kit 2, marked as B; one bottle is left untreated, marked as C. Place three bottles at 37°C, 5% CO 2 Place in the incubator for 30 minutes. 5 minutes before cell inoculation, the coating solution of the culture bottle was aspirated and discarded.

[0035] 2. Use tweezers to take out the umbilical cord from the centrifuge tube and put it into a sterile container. Add umbilical cord washing solution, and fully wash the umbilical cord until the cord blood or dirt on the umbilical cord is washed away.

[0036] 3. Cut the umbilical cord into small pieces of about 3-5cm with scissors. The umbilical vein, artery and amniotic membrane were peeled off from the umbilical cord, and the Wharton's jelly was taken out and placed in a new...

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Abstract

The invention discloses a reagent kit for primary culture on umbilical cord mesenchymal stem cells. The reagent kit comprises umbilical cord cleaning solution, envelope solution and a cell primary culture medium. The umbilical cord cleaning solution is normal saline with penicillin and streptomycin or PBS (phosphate buffer solution) with the pH (potential of hydrogen) of 7.4; the envelope solution is normal saline solution with fibronectin or normal saline solution with type IV collagen; an alpha-MEM culture medium with L-glutamine is used as base solution, and human insulin, transferrin, human serum albumin, vitamin C, trehalose, epidermal growth factors, vascular endothelial growth factors, recombinant human interleukin-3 are added into the base solution to obtain the cell primary culture medium which is culture solution. The reagent kit has the advantages that a series of procedures from umbilical cord pretreatment to separation, inoculation and culture can be completely carried out by the aid of the reagent kit, and the reagent kit is convenient to operate and low in cost and is safe and controllable; the cultured umbilical cord MSCs (mesenchymal stem cells) are high in proliferation speed and difficult to age and have broad clinical application prospects.

Description

technical field [0001] The invention relates to a kit for culturing stem cells, in particular to a kit for primary culture of umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with a high degree of self-renewal and multi-directional differentiation potential. Under certain conditions, they can differentiate into fat cells, chondrocytes, osteoblasts, nerve cells, and cardiomyocytes. wait. MSCs originated from the mesoderm and ectoderm, and were first found in the bone marrow. Later studies confirmed that MSCs were also found in fat, muscle, dermis, peripheral blood, umbilical cord blood, and placenta. In addition, MSCs also have the functions of supporting hematopoiesis, immune regulation, and tissue repair. More and more people pay attention to their unique biological characteristics and clinical applications, and now they have become the first choice for cell therapy of various diseases. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 陈莉宋珂慧郭伟张立媛张虎
Owner 济南赛尔生物科技股份有限公司
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